Virumeter for rapid detection of covid19 and other pathogens

ABSTRACT

Embodiments may include a virumeter for rapid detection of COVID-19 infection and assessment of immunity to the virus. For example, a device for detecting primary antibodies to a pathogen or the pathogen may comprise a cartridge to receive a test sample from the person, the cartridge comprising at least one chamber to receive the test sample, first apparatus to mix at least one first reagent reactive to presence of the primary antibodies to the pathogen or the pathogen, second apparatus to mix at least one second reagent including a fluorescent compound with the test sample reactive to presence of the at least one first reagent having reacted to presence of the primary antibodies to the pathogen or the pathogen, and circuitry to determine presence of primary antibodies to the pathogen or the pathogen by detecting reaction of the second reagent by determining fluorescence of the fluorescent compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/988,320, filed Mar. 11, 2020, U.S. Provisional Application No.62/991,906, filed Mar. 19, 2020, U.S. Provisional Application No.62/993,222, filed Mar. 23, 2020, U.S. Provisional Application No.62/994,165, filed Mar. 24, 2020, and U.S. Provisional Application No.63/001,291, filed Mar. 28, 2020, the contents of which are incorporatedherein in their entirety.

BACKGROUND

The present invention relates to a virumeter that provide rapiddetection of COVID-19 infection and techniques for rapid assessment of aperson's immunity to the virus, for example, using Microscale AffinityChromatography (MAC), indirect ELISA, and optical molecular sensingtechnology.

COVID-19, a disease caused by the novel coronavirus (SARS-CoV-2) thatwas first reported from Wuhan, China, on Dec. 31, 2019, has beendeclared an international pandemic by the World Health Organization. Thevirus is spread between people who are in close contact with one anotherthrough respiratory droplets produced by coughing or sneezing. Theincubation period of COVID-19 is approximately 14 days, making itdifficult to contain and contributing to its rapid spread. Currentdetection techniques rely on RT-PCR, which is a lengthy process,necessitating well-equipped laboratories and skilled personnel toperform the technique. Rapid screening methods are needed to detect thedisease at points of entry, transportation hubs, schools, hospitals, andother areas at high risk for communicating the disease in order to limitthe spread of the virus. Furthermore, rapid methods of assessing aperson's immunity to the virus are becoming increasingly important aspeople plan for returning to schools and workplaces after the peak ofthe epidemic. The need to assess the efficacy of vaccines in developmenthas been recognized and is essential in assessing the “herd immunity” toprevent a resurgence of the pandemic.

Accordingly, a need arises for techniques for rapid detection ofCOVID-19 infection and techniques for rapid assessment of a person'simmunity to the virus.

SUMMARY

Embodiments may include a virumeter that provides rapid detection ofCOVID-19 infection and rapid assessment of a person's immunity to thevirus. For example, embodiments of the present techniques may providerapid, accurate antibody and viral load testing using MicroscaleAffinity Chromatography (MAC), indirect ELISA, and optical molecularsensing technology.

For example, embodiments may provide COVID-19 detection using a markedantibody and fluorescence detection for high sensitivity and fast testtime. Such test may be on the order of seconds or minutes instead ofhours. An embodiments of a test device may be compact and cost effectiveand may not need to be cleaned or serviced between tests. Embodimentsmay include sample cartridges that are pre-filled with the necessarycompounds and are ready to accept a liquid saliva sample for immediatetesting.

In an embodiment, a device for detecting primary antibodies to apathogen or the pathogen in a person may comprise a cartridge configuredto receive a test sample from the person, the cartridge comprising atleast one chamber configured to receive the test sample, first apparatusconfigured to mix at least one first reagent reactive to presence of theprimary antibodies to the pathogen or the pathogen, second apparatusconfigured to mix at least one second reagent including a fluorescentcompound with the test sample reactive to presence of the at least onefirst reagent having reacted to presence of the primary antibodies tothe pathogen or the pathogen, and circuitry configured to determinepresence of primary antibodies to the pathogen or the pathogen bydetecting reaction of the second reagent by determining fluorescence ofthe fluorescent compound.

In embodiments, the first apparatus may comprise a plurality of magneticparticles upon which at least one antigen to primary antibodies to thepathogen has been immobilized, wherein the at least one first reagentcomprises the at least one antigen to primary antibodies that has beenimmobilized on the plurality of magnetic particles. The first apparatusmay further comprise apparatus configured to mix the plurality ofmagnetic particles with the test sample so as to cause the primaryantibodies to the pathogen to attach to the antigen. The secondapparatus may comprise apparatus configured to mix the magneticparticles having the primary antibodies to the pathogen to attachedthereto with the at least one second reagent including a fluorescentcompound. The at least one second reagent including a fluorescentcompound may comprise at least one secondary antibody labeled with afluorescent compound. The circuitry configured to determine presence ofprimary antibodies to the pathogen or the pathogen by detecting reactionof the second reagent by determining fluorescence of the fluorescentcompound may comprise a fluorometer. The circuitry configured todetermine presence of primary antibodies to the pathogen or the pathogenby detecting reaction of the second reagent by determining fluorescenceof the fluorescent compound may comprise a light source configured toexcite the fluorescent compound with a of light and an optical sensorconfigured to detect an emitted spectrum of light from the excitedfluorescent compound. The device may further comprise display circuitryconfigured to display an indication of presence or absence of primaryantibodies to the pathogen. The test sample may be saliva. The pathogenmay be SARS-CoV-2. The antigen may comprise SARS-CoV-2 S1 protein. Thesecondary antibodies may comprise IgA, IgM, and IgG and each of IgA,IgM, and IgG may be labeled with a fluorescent compound having adifferent light emission spectrum. The test sample may comprise saliva,the pathogen is SARS-CoV-2, and the antigen comprises SARS-CoV-2 S1protein.

For example, in an embodiment, a method of detecting primary antibodiesto a pathogen in a person may comprise receiving in a testing device atest sample from the person, mixing with the test sample a plurality ofmagnetic particles upon which at least one antigen to primary antibodieshas been immobilized so as to cause the primary antibodies to thepathogen to attach to the antigen, mixing at least one secondaryantibody labeled with a fluorescent compound with the test sample mixedwith the magnetic particles so as to cause the at least one secondaryantibody to attach to the primary antibodies to the pathogen, anddetermining presence of primary antibodies to the pathogen by detectingsecondary antibody attachment by determining fluorescence of thefluorescent compound.

In embodiments, the test sample may be one of saliva, blood, or a sampleobtained with a nasopharyngeal swab. The pathogen may be SARS-CoV-2. Theantigen may comprise SARS-CoV-2 S1 protein. The fluorescence of thefluorescent compound may be detected using a fluorometer. Thefluorescence of the fluorescent compound may be determined by excitingthe fluorescent compound with a spectrum of light, and detecting emittedlight from the excited fluorescent compound. The secondary antibodiesmay comprise IgA, IgM, and IgG and each of IgA, IgM, and IgG is labeledwith a fluorescent compound having a different light emission spectrum.The test sample may comprise saliva, the pathogen may be SARS-CoV-2, andthe antigen may comprise SARS-CoV-2 S1 protein.

In an embodiment, an apparatus for detecting primary antibodies to apathogen in a person may comprise a cartridge configured to receive atest sample from the person, the cartridge comprising at least onechamber configured to receive the test sample and containing a pluralityof magnetic particles upon which at least one antigen to primaryantibodies has been immobilized, and configured to mix the plurality ofmagnetic particles with the test sample so as to cause the primaryantibodies to the pathogen to attach to the antigen, apparatusconfigured to move the magnetic particles to at least one chamber havingat least one secondary antibody labeled with a fluorescent compound, andto mix the at least one secondary antibody with the magnetic particlesso as to cause the at least one secondary antibody to attach to theprimary antibodies to the pathogen, and circuitry configured todetermine presence of primary antibodies to the pathogen by detectingsecondary antibody attachment by determining fluorescence of thefluorescent compound.

In embodiments, the test sample may be one of saliva, blood, or a sampleobtained with a nasopharyngeal swab. The pathogen may be SARS-CoV-2. Theantigen may comprise SARS-CoV-2 S1 protein. The circuitry configured todetermine the presence of primary antibodies to the pathogen bydetecting secondary antibody attachment by determining fluorescence ofthe fluorescent compound may comprise a fluorometer. The circuitryconfigured to determine the presence of primary antibodies to thepathogen by detecting secondary antibody attachment by determiningfluorescence of the fluorescent compound may comprises a light sourceconfigured to excite the fluorescent compound with a spectrum of light,and an optical sensor configured to detect emitted light from theexcited fluorescent compound. The secondary antibodies may comprise IgA,IgM, and IgG and each of IgA, IgM, and IgG is labeled with a fluorescentcompound having a different light emission spectrum. The test sample maycomprise saliva, the pathogen is SARS-CoV-2, and the antigen maycomprise SARS-CoV-2 S1 protein.

BRIEF DESCRIPTION OF THE DRAWINGS

The details of the present invention, both as to its structure andoperation, can best be understood by referring to the accompanyingdrawings, in which like reference numbers and designations refer to likeelements.

FIG. 1 is an exemplary schematic representation of viral detection.according to embodiments of the present techniques.

FIG. 2 are exemplary schematic representations of target antibodyimmobilization on a surface according to embodiments of the presenttechniques.

FIG. 3 is an exemplary block diagram of Microscale AffinityChromatography (MAC) according to embodiments of the present techniques.

FIG. 4 is an exemplary schematic representation of optical detectionaccording to embodiments of the present techniques.

FIG. 5 is an exemplary schematic representation of optical detectionaccording to embodiments of the present techniques.

FIG. 6 is an exemplary flow diagram of a testing process according toembodiments of the present techniques.

FIG. 7a is an exemplary top view of sample test cartridge according toembodiments of the present techniques.

FIG. 7b is an exemplary bottom view of sample test cartridge accordingto embodiments of the present techniques.

FIG. 8 is an exemplary illustration of a cartridge movement apparatusaccording to embodiments of the present techniques.

FIG. 9 is an exemplary illustration of an embodiment of fluorescencedetection according to embodiments of the present techniques.

FIG. 10 is an exemplary block diagram of a test cartridge according toembodiments of the present techniques.

FIG. 11 is an exemplary block diagram of a testing device according toembodiments of the present techniques.

FIG. 12a is an exemplary illustration of a front panel of a testingdevice according to embodiments of the present techniques.

FIG. 12b is an exemplary illustration of a front panel of a testingdevice according to embodiments of the present techniques.

FIG. 13a is an exemplary internal view of a testing device according toembodiments of the present techniques.

FIG. 13b is an exemplary internal view of a testing device according toembodiments of the present techniques.

FIG. 14 is an exemplary block diagram of a testing system according toembodiments of the present techniques.

FIG. 15 is an exemplary block diagram of a computing device, in whichprocesses involved in the embodiments described herein may beimplemented.

FIG. 16 is an exemplary block illustration of antibody types present atdifferent stages of disease, as may be utilized by embodiments of thepresent techniques.

FIG. 17 is an exemplary internal view of a testing device according toembodiments of the present techniques.

FIG. 18 is an exemplary block diagram of a process of flow-basedsandwich immunoassay according to embodiments of the present techniques.

DETAILED DESCRIPTION

Embodiments may include techniques that provide, rapid detection ofCOVID-19 infection and techniques for rapid assessment of a person'simmunity to the virus. For example, embodiments of the presenttechniques may provide rapid, accurate antibody and viral load testingusing Microscale Affinity Chromatography (MAC), indirect ELISA, andoptical molecular sensing technology.

Due to the nature of the coronavirus and its ability to spread quickly,there is an immediate need for a portable, instant, non-invasive testthat does not require skilled technicians or lab equipment. Currently,only patients who experience severe symptoms are tested in hospitalsbecause tests are too expensive and short in supply. Meanwhile,unscreened patients who have mild or no symptoms continue their dailylife, increasing the scale of contamination. The current gold standardfor SARS-CoV-2 detection is real time RT-PCR (reversetranscription-polymerase chain reaction). The drawbacks of RT-PCR aremulti-faceted in that the equipment is expensive, conducting testsrequires expertise, and results take hours to acquire. A completereaction can be performed in as little as 4 hours, however, thecollection of samples, transportation to the lab, preparation ofequipment and analysis of results mean significantly longer time isrequired. Increasing demand for samples to be tested, along with alimited supply of reagents, skilled staff and equipment, can extend theentire RT-PCR process from sample collection to final result to severaldays. Furthermore, not only is RT-PCR time consuming (average turnaround3-6 days), but it is also highly labor and cost intensive chargingpatients and insurance companies up to $4,000 per test. Therefore, thereis a recognized need for more rapid, inexpensive tests.

More rapid diagnostic tests have recently come to the market. However,these tests lack reproducibility and have a higher risk of providingfalse positives. These technologies also utilize forms of sampling thatrequire proximity to the patient, leaving healthcare workers at a higherrisk of contracting the disease, such as a nasotracheal swab or a bloodsample. The risk to healthcare workers can be minimized by simplyacquiring a pooled saliva sample, as SARS-CoV-2 has been detected insaliva of infected patients.

Additionally, evidence shows that convalescent plasma from patients whohave recovered from viral infections can be used as a treatment forinfection without severe adverse events. The rationale behindconvalescent plasma therapy is that the antibodies from a recoveredpatient's serum might suppress viraemia in an ill patient. The abilityto quickly screen recovered patients and administer their convalescentplasma to ill patients is an area lacking in proper diagnostic tests.

After the threat of the pandemic has been mitigated, a need to screenthe population to assess the efficacy of vaccines towards the virus tobuild “herd immunity” has been recognized. Herd immunity (also calledcommunity immunity) is an important mechanism by which the largercommunity is protected. For some diseases, if enough people are immune,transmission of the disease is reduced or eliminated. In the case ofprotecting against a resurgence of another global pandemic, herdimmunity will need to be assessed to determine vulnerable sites with thepotential to become hotspots.

Embodiments of the present techniques may include a process to rapidlydetect COVID-19 through an alternative method of testing crude salivasamples. Embodiments may utilize lab-on-a-chip technology, where apatient sample containing saliva may be analyzed for antibodiescapturing the virus with a simultaneous release of a fluorescent markerligand. The lab on a chip technology may have the capability to producea read out in real time using a fluorescent marker passing through afluorometer. The lab on a chip technology may provide reusablecapability, allowing for multiple testing opportunities from one device.

Embodiments may provide a completely self-contained device, withsingle-use sampling chips, which will reduce the risk of carry-over andminimize error as the sample does not have to be handled after it isloaded. All reagents needed for buffering, dilutions, or washing may befully contained in a multiple use cartridge, which can be replaced orrefilled as needed. Embodiments may be utilized at transportation hubs,such as airports, schools, clinics, and hospitals to limit the spread ofthe virus, and the technology may be expanded to screen for immunitytowards other infectious diseases. In embodiments, the sampling chipsand devices may be designed to concurrently detect the presence of bothantibodies and virus in the same sample by utilizing the methodsdescribed below on the same sampling chip and in the same device.

In embodiments, the testing device may be an automated sandwichimmunoassay with fluorescence detection intended for detection of IgA,IgM, IgG antibodies toward SARS-CoV-2 in patients with an active immuneresponse to the virus. Results may identify an immune response toSARS-CoV-2. The screened antibodies are generally detectable in salivaduring all phases of infection. Positive results from the embodiments ofthe testing device are indicative of an active immune response to thevirus, that is, that an individual has been infected with SARS-CoV-2 andfacilitated a targeted immune response to the virus.

In embodiments, the testing device is intended for use by consumers,clinicians, and point-of-care facilities. In embodiments, the testingdevice may be used by a variety of consumers in both traditionalhealthcare settings and elsewhere, for example, in clinics andhospitals, point of entry locations, and private settings, such as athome and in commercial locations. A trained technician is not requiredto operate the device, and results can easily be read by both licensedhealthcare professionals and private individuals. Such accesssignificantly increases the likelihood to determine transmission,resulting on an immediate impact on local and global health.

Embodiments may include a self-contained device that may utilizesingle-use disposable test cartridges, containing microfluidic chips.Each disposable test cartridge may be self-contained, and the sample maybe magnetically moved through the cartridge's microfluidic chip. Thetest cartridge comes with all necessary reagents, and cleaning suppliesare not applicable due to the disposable nature of the cartridges.Additional testing cartridges can be ordered as needed.

Embodiments may include a testing device utilizing automated sandwichimmunoassay with fluorescence detection in a microfluidic device for thedetection of anti-SARS-CoV-2 IgA, IgM, and/or IgG antibodies in salivasamples. S1 protein from the SARS-CoV-2 virus or a mixture of proteinsor their subunits from the virus may be immobilized on, for example,magnetic particles. The magnetic particles may be moved through the useof electromagnets to mix with the saliva sample. In embodiments,magnetic particles that may be used may include, for example, EpoxySilica Magnetic Particles, 6 μm, Ni-NTA Silica Magnetic Particles, 6 μm,etc.

Embodiments may use the principle of a sandwich immunoassay on magneticbeads. Spike protein specific to the SARS-CoV-2 virus or a subunit ofthis protein may be immobilized on the surface of silica-coated magneticbeads. Anti-SARS-CoV-2 IgA, IgM, or IgG antibodies from the salivasample will attach to the antigen immobilized on these beads. Examplesof the antigen may include a recombinant SARS-CoV-2 spike protein or thes1 subunit of the spike protein, etc., these proteins may be his-tagged.The magnetic particles will be moved through the cartridge to mix withsecondary antibodies labeled with a fluorescent molecule. Examples offluorescent molecules may include QUANTABLU™ Fluorogenic Substrate,having an excitation maximum at about 325 nm and an emission maximum atabout 420 nm, QUANTARED™ Enhanced Chemifluorescent Substrate having anexcitation maximum at about 570 nm and an emission maximum at about 585nm, fluorescein: having an excitation maximum in a range of about475-495 nm and an emission maximum in a range of about 510-520 nm, etc.The anti-IgA, anti-IgM, and anti-IgG secondary antibodies will serve todetect the binding of antibodies from a positive sample to theimmobilized spike protein. Examples of antibodies may include anti-GoatIgG, anti-Goat IgG-FITC labeled, anti-human IgG, anti-human IgM,anti-rabbit IgG, anti-rabbit IgG-FITC labeled, etc. After a wash stepwith a neutral buffer, to remove excess antibodies, the beads may bemoved under a fluorescence detector in the device to detect the signal.Examples of buffers may include tris-buffer, pH ˜7 (for wash), phosphatebuffer, pH ˜7 (for wash), etc. A solution of 10 N HCl may be used forregeneration of the cartridge if desired.

The antibodies raised against SARS-CoV-2 that are present in the salivafrom patients with an active immune response to the virus will bind tothe immobilized antigen on the particles. The magnetic particles willthen be mixed with a fluorescent-labeled secondary antibody, which willbind to anti-virus antibodies, which were present in the sample. Inembodiments, the magnetic particles will then be passed into a washingarea, then passed under a fluorescent detector, which will detect thesignal.

In embodiments, the results may be indicated by a colored light, such asgreen, yellow or red. Green indicates a positive result, meaninganti-SARS-CoV-2 antibodies have been detected at or above a level thatrepresents current or past infection by SARS-CoV-2 and an active immuneresponse. Yellow indicates an indeterminate result (i.e., user error),and red indicates a negative result, meaning that no anti-SARS-CoV-2antibodies are present.

In embodiments, testing device capacity may be, for example, severalhundred tests per day. In embodiments, the total time required toperform the test may be, for example, five (5) minutes. In embodiments,the number of tests that can be performed per testing device may be oneper run.

In embodiments, the estimated shelf life of reagents may beapproximately six (6) months with refrigeration. Without refrigeration,shelf life is expected to be shortened, but still likely to be suitablefor intermediate to long-term use, for example, approximately three (3)to six (6) months. In embodiments, nM concentrations of antibodies maybe used, which are typical concentrations for enzyme based assays.Specific materials to be used may include IgA, IgM, and/or IgGantibodies. Cross-reactivity with other pathogens or antibodies towardsother pathogens is not expected. The S1 subunit of the SARS-CoV-2 virusis expected to react specifically with antibodies against the virus.

Detection of Antibody. Embodiments may provide a rapid COVID-19 screenthat utilizes a sandwich assay or indirect enzyme-linked immunosorbentassay (ELISA) method. A sandwich immunoassay is a method using twoantibodies, which bind to different sites on the antigen or ligand, asshown in FIG. 1. The capture antibody, which is highly specific for theantigen, is attached to a solid surface. The antigen is then added,followed by addition of a second antibody referred to as the detectionantibody. The detection antibody binds the antigen at a differentepitope than the capture antibody. As a result, the antigen is‘sandwiched’ between the two antibodies.

An overview of a flow-based sandwich immunoassay process 1800 accordingto the present systems and methods is shown in FIG. 18. In process 1800,a Sandwich Immunoassay may be conducted based on two antibodies andbinding agents to measure a target compound located in the cartridge ofthe device. At 1802, a sample may be sample with a first binding agenton a support in a flow-based system. For example, The first bindingagent, a COVID-19 (SARS-CoV-2) specific antigen, may be attached tomagnetic beads and may be used to capture the anti-COVID-19 IgA, IgM,and IgG antibodies from the saliva sample. At 1804, one or moresecondary labeled antibodies may be added (this may instead be done at1082). Each secondary antibody may contain a fluorescent label that willbe used to detect and measure the amount of the capture agent. At 1806,labeled antibodies may be detected, either on the support or after theirrelease with an elution buffer. This approach is highly advantageous asit allows for the detection of any stage of infection(early/late/resolved) by identifying at least three isotypes ofantibodies.

As shown in FIG. 1, a crude saliva sample 102 may be injected into acapillary 104 etched onto a silica microfluidic chip 106. For example,saliva may be collected into a 1 mL sterile tube, such as an Eppendorftube, and transferred with a sterile plastic Pasteur pipette, which willbe included with the cartridge. This device is already FDA cleared andwidely available. In embodiments, once the cartridge with the salivasample is put into the device, the remaining steps may be performedautomatically by the device. The anti-SARS-CoV-2 antibodies in thesample will be selectively separated by the magnetic beads with spikeprotein antigen attached. Any particulate matter will be left behind inthe initial port where the sample is introduced. In embodiments, thereis no sample preparation that needs to be done by the user.

Capillary 104 contains an immobilized SARS-CoV-2 specific antigen 108 todetect SARS-CoV-2 specific antibodies in the sample. If a samplecontains antibodies towards the virus, indicating an immune response hasoccurred, these antibodies 110 will attach to the immobilized antigen.After the sample is run through the capillary, a detection reagentcontaining a secondary antibody 112 tagged with a fluorescentfluorophore 114 will be introduced. Secondary antibody 112 willrecognize blood or saliva-borne antibodies 110 which have attached toimmobilized antigen 108. A fluorometer (not shown) will then detectwhether secondary antibody binding has occurred. A fluorometer may beused to measure parameters of visible spectrum fluorescence such as itsintensity and wavelength distribution of emission spectrum afterexcitation by a certain spectrum of light. This is described furtherbelow with reference to FIG. 4.

This approach is highly advantageous as it allows for the detection ofany stage of infection (early, late or resolved) by identifying threeisotypes of antibodies.

Immobilization of Antigen. Embodiments may include disposable silicachips 106 be pre-packaged with SARS-CoV-2 specific antigen 108,preferably the S1 protein, directly immobilized onto etched capillariesor onto magnetic particles or beads. Antigen will be diluted in bindingsolution (0.2 M carbonate-bicarbonate), added to the chip, andincubated. Deactivated surfaces will be used to prevent the need for ablocking step to prevent the non-specific binding of antibodies to thechip. The user need only collect a crude saliva sample, which can thenbe introduced to the column for detection.

Use of Saliva Sample. Not only is the use of pooled saliva less invasivethan blood or nasopharyngeal swabs, but it also minimizes exposure forhealthcare workers. Some virus strains have been detected in saliva aslong as 29 days after infection. SARS-CoV-2 can present in the saliva inat least three ways. First, SARS-CoV-2 in the lower and upperrespiratory tract can enter the oral cavity with the liquid dropletsfrequently exchanged by these organs. Second, SARS-CoV-2 in the bloodcan access the mouth via crevicular fluid. Third, major- andminor-salivary gland infection, with subsequent release of SARS-CoV-2particles in saliva via salivary ducts can cause SARS-CoV-2 to presentin the saliva.

Recent tests for other viral diseases, such as HIV, are employing salivain a similar fashion as they have advantages over blood-based tests interms of quality, rapidity and convenience. The sensitivity,specificity, positive predictive value and negative predictive value ofsuch HIV tests may be quite high.

Pooled saliva samples can be used to detect both IgG and IgM antibodies,which pass into the mouth through the mucosa, and IgA which are secretedin the mouth. The production of IgM, IgA and IgG antibodies againstCOVID-19 were found in patient serum as early as day 1 after symptomonset. IgA antibodies were detected in 92.7% of patient samplescollected within 0-7 days of symptom onset. IgM and IgA antibodies wereboth detectable at day 5, and the detection time of IgM, IgA, and IgGagainst COVID-19 ranged from day 1 to 39 PSO.

One study found the sensitivity and specificity of a lateral flow kitutilizing blood to detect COVID-19 IgM and IgG was 88.66% and 90.63%,respectively (Li, Z., et al., 2020). As the present techniques willadditionally test IgA, and since the profile of antibodies in the salivais similar to that in blood, embodiments of the present techniquesshould yield similar, if not higher, sensitivity and specificity, aswell as a higher accuracy in detecting patients at any stage ofinfection.

Detection of Antibody. Along with the crude saliva sample, a detectionreagent including a secondary antibody labeled with a fluorophore willbe introduced to the chip. In embodiments, non-captured samplecomponents and any non-bound secondary antibody can be effectivelywashed from the device using an application buffer, with the possibleuse of additives to minimize non-specific binding, as antibodies againstthe viral antigen are captured by the support. Alternatively, inembodiments, the sample may be applied first to the support, followed byapplication of the labeled secondary antibodies, with the non-capturedor non-bound components again being washed from the support during thisprocess. The conjugated antibodies that are used for this process may beobtained from existing sources or prepared according to well-establishedprocedures for adding fluorescent tags to antibodies or other secondarybinding agents. The performance of this device over extended use can bemonitored by analyzing positive and negative control samples along withsamples.

In addition to detecting immunity, embodiments may be used to verifyvaccine immunogenicity. Embodiments may assist researchers in assessingwhether the correct antibody profile necessary to protect a patient fromreinfection is present. The overall estimated effectiveness of seasonalinfluenza vaccine for preventing medically attended,laboratory-confirmed influenza virus infection in the 2019 to 2020 fluseason was only 45% (Dawood, F. S., et al.). In clinical trials, thismethod can be used to confirm that antibodies have been raised to thepathogen, meaning the patient has had effective coverage from infectionwith the pathogen, and has been applied in confirming the effectivenessof the rabies vaccine in dogs and cat (Servat, A., et al., 2007). Inaddition, immunity to multiple diseases can be screened using a tunablelaser and different fluorescent markers.

Rapid COVID-19 Viral Detection. Embodiments may combine viral detectionand antibody detection onto one device, or embodiments may providestand-alone viral or antibody detection product depending on the needsof the market. In embodiments, a rapid COVID-19 viral detection test mayemploy Microscale Affinity Chromatography (MAC) technology, a separationtechnique that combines the specificity of antibody recognition andbinding with the power, efficiency and speed of modern liquid-phaseseparations. An example of Microscale Affinity Chromatography 300,according to embodiments of the present techniques, is shown in FIG. 3.A saliva sample 302 is loaded 304 onto a silica microfluidic chamber 306etched with columns 308 on which primary antibodies 309 towardsSARS-CoV-2 are immobilized. Low-affinity fluorophores 310 are weaklybound to these antibodies and are displaced 312 when a sample containingSARS-CoV-2 is introduced. Displaced fluorophores 312 can be consideredan indication of a positive result.

In embodiments, antibodies 309 towards SARS-CoV-2 specific proteins willbe immobilized on the surface of micro-capillaries 308, microcolumns oretched silica channels on a lab-on-a-chip technology and then taggedwith a low affinity competitive ligand 310 containing a fluorophore. Acrude patient sample 302, such as saliva, oral or nasopharyngeal swab orblood, can be introduced onto the channel 308 and migrated through thedevice either by electric charge or a flow system. If SARS-CoV-2 ispresent in the sample, the virus antigens will be captured on thecolumns 312 by the antibodies 309 releasing the low affinity ligand 310to the end of the column. The viral antigens in sample 302 have a higheraffinity or liking for the antibodies 309 and therefore will bind to theantibodies 309 and elute the ligand 310 which has a lower affinity. Ifthe low affinity ligands 310 are released from the antibodies 309, afluorescent marker ligand 310 will be released to indicate a positivesample. If no fluorescence is seen, the sample is negative, because thelow affinity ligand 310 was never released from the antibodies.

Sampling. Due to ease of sampling, and lack of necessity for samplepreparation, embodiments may use a crude saliva sample 302. The subjectwill spit into a tube, and a plastic pipette or dropper may be used totransfer the saliva to the etched chip 306. Embodiments may usealternative sampling techniques, such as a nasopharyngeal swab, oralswab or blood sample. In embodiments, a breathalyzer may be interfacedwith the COVID-19 detection device. In embodiments, samples fromsurfaces or air may also be tested using swab methods, or air samplingmethods, respectively.

Although the example of Microscale Affinity Chromatography shown in FIG.3 is described in terms of detection of SARS-CoV-2, the describedtechniques and apparatus are equally applicable to detection of otherpathogens, such as viruses, bacteria, etc.

Antibody Immobilization to Silica Chip. Exemplary schematicrepresentations of target antibody immobilization on surface, such asgold, using (a) direct target antibody 202, (b) protein A/G-mediated204, and (c) secondary antibody-mediated immobilization strategies 206are shown in FIG. 2. In embodiments, antibodies can be attached directly202 to the walls of a capillary column or microchip channel, however,the orientation of the stationary antibody is key to the bindingactivity. Antibodies can be immobilized covalently, using a thiol group208, to a surface or connected to a solid support, although the orientedimmobilization of antibodies is considered to be optimal for theireffectiveness. An antibody is considered to be properly oriented andperfectly active 210 when the fragment crystallizable region (Fc), whichhas no antigen binding affinity, is immobilized on a surface, ratherthan the antigen-binding sites 214 being immobilized on the surface 212.This situation can be produced by a covalent immobilization method, suchas carbohydrate groups in an antibody's Fc region. Directly immobilizedantibodies do not allow for specific orientation of the antibody, thus,embodiments may use other methods to immobilize antibodies to a silicamicrofluidic chip, as described below.

It is also possible to achieve proper immobilization through secondarymolecule protein A/G-mediated immobilization 204, or secondaryAb-mediated immobilization 206. In protein A/G-mediated immobilization204, the biomolecules used for antibody immobilization are proteins Aand G 216. Protein A is the most successful surface protein able to bindwith animal immunoglobulin G (IgGs), but is not effective in certainanimal IgGs, such as goat, sheep, cow, and horse. Protein G reacts morewith IgGs than protein A and reacts less with other antibody types. Arecombinant protein A/G that combines four immunoglobulin-bindingdomains from protein A and two from protein G can be employed to modifysilane-functionalized silicon nitride surfaces.

In Ab-mediated immobilization 206, secondary antibodies 218 are attachedto the support and used to recognize the Fc region of the primaryantibodies against the target. In this situation, the binding ability ofthe secondary antibodies should match with the class or subclass of theprimary antibody that is to be used immobilized. For example, if theprimary antibody is one of mouse IgG subclasses or rabbit IgG, ananti-mouse IgG or anti-rabbit IgG could be used as secondary antibodies.After immobilizing the thiolated-secondary antibody on a silica surface,the target antibody can be captured by the secondary antibody in thecorrect orientation by binding between the Fab region of the secondaryantibody and the Fc region of target antibody.

The example of target antibody immobilization on surface shown in FIG. 2is not described in terms of detection of any particular pathogen.Rather, the described techniques and apparatus are applicable todetection of many pathogens, such as viruses, such as SARS-CoV-2,bacteria, etc.

Primary Antibody. There are 4 conserved structural proteins across CoVs:the spike (S) protein, membrane (M) protein, envelope (E) protein, andnucleocapsid (N) protein. The S protein is responsible for binding tohost cell receptors and viral entry to host cells. The M, E, and Nproteins are part of the nucleocapsid of viral particles. S and N genesare under episodic selection as the virus is transmitted between humans.Mutations and adaptation in the S and N genes may affect virus stabilityand pathogenicity.

Embodiments may use a single primary antibody towards a conservedportion of any of the aforementioned proteins can be used, orembodiments may use a mixture of primary antibodies, which are specificto different mutations of the virus. Monoclonal antibodies towards aconserved portion of the S1 spike surface proteins of SARS-CoV-2 may bea primary target of embodiments. However S2, M, E, or N antibodies maybe tested in embodiments.

An example of Microscale Affinity Chromatography with CompetitiveAffinity Ligand and optical sensing 400, according to embodiments of thepresent techniques, is shown in FIG. 4. A saliva sample 402 is loaded404 onto a silica microfluidic chamber 406 etched with columns 408 onwhich primary antibodies 410 towards SARS-CoV-2 are immobilized. Inembodiments, a low-to-moderate affinity, competitive ligand fluorophore412 will be attached to the primary antibody 410 before the sample 402is run through the column 408. This fluorescent compound will attach tothe primary antibody immobilized on the chip, as visualized in FIG. 2.As the sample 402 is run 414 through the column 408, SARS-CoV-2 withinthe sample will compete for binding to the primary antibody 410 and,because it has a higher affinity towards the antibody 410, will displacethe fluorophore 412. The displaced fluorophore 412 will then be used asan indication of a positive result, and lack thereof can be regarded asa negative result. The concentrated sample may then be eluted 416 foranalysis.

In embodiments, fluorescein, a xanthene dye that is highly fluorescent,and detectable even when present in minute quantities may be used.Embodiments may use one of numerous fluorescent markers that areavailable to serve this function. For example, fluorescein has anexcitation maximum in a range of about 475-495 nm and emission in arange of about 510-520 nm, QUANTABLU™ Fluorogenic Substrate, has anexcitation maximum at about 325 nm and an emission maximum at about 420nm, QUANTARED™ Enhanced Chemifluorescent Substrate having an excitationmaximum at about 570 nm and an emission maximum at about 585 nm, etc.

Rapid Optical Detection and Improved Sensing. In embodiments, after theconcentrated sample containing the biomolecules of interest is eluted416, additional analysis may be performed using a novel lab-on-chiputilizing wavelength backscattering with at least two wavelengths oflight. For example, a light source 418 capable of emitting at least twowavelengths of light, such as a plurality of light emitting diodes,laser diodes, or other lasers, or a tunable laser, may be used toilluminate the eluted sample, exciting the fluorophore 412 and causinglight emission 420. The emission spectrum of the light emitted 420 fromthe fluorophore 412 may be optically sensed 420 and analyzed by, forexample, an optical sensing “box” or circuit 422. Optical sensingcircuit 422 may perform wavelength and amplitude analysis on the emittedlight 420 and may determine the absence, presence, and/or quantity ofSARS-CoV-2, or other pathogen, or antibody in the sample 402. Inembodiments, such determination may be made in optical sensing circuit422 and communicated to a computing device 426, such as a smartphone,tablet computer, laptop computer, personal computer, workstationcomputer, cloud computing service, etc. In embodiments, optical sensingcircuit 422 generate data representing the performed wavelength andamplitude analysis and may transmit that data to a computing device 426,such as a smartphone, tablet computer, laptop computer, personalcomputer, workstation computer, cloud computing service, etc., fordetermination of the absence, presence, and/or quantity of SARS-CoV-2,or other pathogen, or antibody in the sample 402.

An example of an embodiment of detection of antibodies raised againstSARS-CoV-2 with optical sensing 500, according to embodiments of thepresent techniques, is shown in FIG. 5. It is best viewed in conjunctionwith FIG. 6, which is a flow diagram of an embodiment of the testingprocess 600. Process 600 begins with 602, in which a saliva sample 502may be collected. The specimen volume may, for example, be less than 1mL. For example, saliva may be collected into a 1 mL sterile tube, suchas an Eppendorf tube, and transferred with a sterile plastic Pasteurpipette. In embodiments, once the cartridge with the saliva sample isput into the device, the remaining steps may be performed automaticallyby the device.

At 604, particulate matter may be removed from the sample 502 duringsample preparation. In embodiments, the anti-SARS-CoV-2 antibodies inthe sample will be selectively separated by the magnetic beads withspike protein antigen attached. Any particulate matter will be leftbehind in the initial port where the sample is introduced. Inembodiments, there is no sample preparation that needs to be done by theuser. At 606, a portion of saliva sample 502 may be introduced 504 intothe cartridge loaded 506 including at least one silica microfluidicchamber 508. For example, about 20-100 μL of the sample 502 may beintroduced 504 into the cartridge 506. In embodiments, only a crudeapproximation of the sample and insertion of the sample by a steriledisposable pipette is necessary. The device may only accept a controlledamount of sample (˜30 μL), so the amount of sample measured by the userand inserted into the device need to be exact. The pipette may collectat least 100 μL, which is more than needed. The excess saliva may bediscarded with the pipette.

At 608, cartridge 506 may be inserted into the device, and magneticparticles 510 in the cartridge may be moved to the sample in chamber 508and mixed 512 with the sample. Magnetic particles 510 may have one ormore antigens 514 to antibodies raised against SARS-CoV-2 immobilized onthe particles. Antibodies present in the sample will bind to theantigens 514 immobilized onto magnetic particles 510. Magnetic particles510 may be moved by application of electric current by testing devicecircuitry to magnetic coils. In embodiments, the magnetic coils may beformed on cartridge 506. In embodiments, the magnetic coils may bepresent in the test device and may be adjacent to or in the vicinity ofcartridge 506.

At 610, secondary antibodies (such as IgA, IgM, and IgG) 516 labeledwith a fluorescent compound 518, such as fluorescein, QUANTABLU™,QUANTARED™, etc., will also be combined 520 with the magnetic particlesand mixed to detect captured antibodies from the sample. At 612, themagnetic particles 510 with attached IgA, IgM, and IgG 516 andfluorescent compound 518, together indicates as 522, may be moved to awashing station 524, and washed 526 using a neutral buffer. At 614, themagnetic particles, etc. 522 may be moved to a detection region 528 toobtain the signal 530 from the fluorescent compound 518 labelling thesecondary antibodies 516 on the magnetic particles 510. Antibodyisotypes may be distinguished using the color or light emission spectrumof fluorescent compound attached to the secondary antibody.

In embodiments, fluorescein, a xanthene dye that is highly fluorescent,and detectable even when present in minute quantities may be used.Embodiments may use one of numerous fluorescent markers that areavailable to serve this function. For example, fluorescein has anexcitation maximum in a range of about 475-495 nm and emission in arange of about 510-520 nm, QUANTABLU™ Fluorogenic Substrate, has anexcitation maximum at about 325 nm and an emission maximum at about 420nm, QUANTARED™ Enhanced Chemifluorescent Substrate having an excitationmaximum at about 570 nm and an emission maximum at about 585 nm, etc.

In embodiments, analysis may be performed using a novel lab-on-chiputilizing wavelength backscattering with at least two wavelengths oflight. For example, a light source 532 capable of emitting at least twowavelengths of light, such as a plurality of light emitting diodes,laser diodes, or other lasers, or a tunable laser, may be used toilluminate the washed sample 522, exciting the fluorophore 518 andcausing light emission 530. The emission spectrum of the light emitted530 from the fluorophore 518 may be optically sensed 534 and analyzedby, for example, an optical sensing “box” or circuit 536. Opticalsensing circuit 536 may perform wavelength and amplitude analysis on theemitted light 530 and may determine the absence, presence, and/orquantity and isotype of antibody in the sample 522. In embodiments, suchdetermination may be made in optical sensing circuit 536 andcommunicated to a computing device 538, such as a smartphone, tabletcomputer, laptop computer, personal computer, workstation computer,cloud computing service, etc. In embodiments, optical sensing circuit536 may generate data representing the performed wavelength andamplitude analysis and may transmit that data to a computing device 538,such as a smartphone, tablet computer, laptop computer, personalcomputer, workstation computer, cloud computing service, etc., fordetermination of the absence, presence, and/or quantity of antibody inthe sample 522.

In embodiments, no interpretation of results is needed by the user. Theresponse may be given by a green, yellow, or red indicator per antibodyisotype. Green may indicate a positive result, meaning the antibody hasbeen detected against SARS-CoV-2, representing current or past infectionby SARS-CoV-2 and an active immune response. An indeterminate orinconclusive result may be shown with a yellow indicator, which islikely due to user error or device malfunction. Red may indicate anegative result, meaning no immune response to SARS-CoV-2 was detected.

In embodiments, the testing device may be a self-contained device thatdoes not require a laboratory for interpretation of results. Inembodiments, the positive and negative controls may either be includedwith the device for consumer use or may be directly built into theself-contained device. For example, a separate channel with magneticparticles labeled with a fluorescent molecule can act as an internalcontrol, which will be directly built into the disposable testingcartridge.

Because each testing cartridge is a single use device, representativecartridges from a given batch may be tested with positive or negativecontrols and if these are found to be valid and acceptable, othercartridges in the batch may be used. This process can be performedperiodically to confirm the validity of the devices in the same batch.The testing device itself may be tested using special-purposecartridges. Different types of cartridges may have differentauthentication chips embedded in them, allowing the device to work in“live” or “testing” mode as required. Such authentication chips mayinclude authentication circuitry, identification circuitry, data storagecircuitry, etc., and may identify the type of chip, the type of testingbeing performed, the patient being tested, etc.

An example of a test cartridge 700 is shown in FIGS. 7a and 7b . Anexemplary top view of sample test cartridge 700 is shown in FIG. 7a . Inthis example, test cartridge 700 may include inlet port 702, mixerchamber 704, wash chamber 706, optional amplification chamber 708, mixerreservoir 712, wash reservoir 714, and optional amplification reservoir716. Reservoirs 712, 714, and 716 may contain solvents, reagents,buffers, etc., in hermitically sealed blisters for storage. Mixerreservoir 712 may be pre-filled with the necessary reagent solvent andmicromagnetic beads or particles having antigens to antibodies raisedagainst SARS-CoV-2 immobilized on the particles. Wash reservoir 10041005 may be pre-filled with buffer agent. Optional amplificationreservoir 716 may be prefilled with enzymatic amplification agents orbuffer. Test cartridge 700 may also include a plurality of passages,such as passage 718 between inlet port 702 and chamber 704, passage 720between chamber 704 and chamber 706, and passage 722 between chamber 706and chamber 708.

In operation, the testing device may process test cartridge 702, forexample, as described in conjunction with FIG. 6. Process 600 beginswith 602, in which a saliva sample may be collected. At 606, a portionof saliva sample may be introduced into via inlet port 702 into chamber704. At 608, cartridge 702 may be inserted into the device, and magneticparticles or beads in chamber 706 may be mixed with the sample. Themagnetic particles may have one or more antigens to antibodies raisedagainst SARS-CoV-2 immobilized on the particles. Antibodies present inthe sample will bind to the antigens immobilized onto magneticparticles. Mixing may be facilitated by movement of the magneticparticles. The magnetic particles may be moved by application ofelectric current by testing device circuitry to magnetic coils or bymovement of a magnetic field produced by magnetic coils or by apermanent magnet relative to cartridge 702, as described below.

At 610, a reagent solvent in chamber 712, including secondary antibodies(such as IgA, IgM, and IgG) labeled with a fluorescent compound, such asfluorescein, QUANTABLU™, QUANTARED™, etc., may be combined with themagnetic particles and mixed in chamber 706 to detect capturedantibodies from the sample. At 612, the magnetic particles with attachedIgA, IgM, and IgG and fluorescent compound may be moved to a washingstation in chamber 706, and washed using a neutral buffer from chamber714. At 613, the magnetic particles may optionally be moved intooptional enzymatic amplification chamber 708 and optionally mixed withenzymatic amplification agents or buffer from optional amplificationreservoir 716. At 614, the magnetic particles may be moved to adetection region in chamber 710 to obtain the signal from thefluorescent compound labelling the secondary antibodies 516 on themagnetic particles. Antibody isotypes may be distinguished using thecolor or light emission spectrum of fluorescent compound attached to thesecondary antibody.

An exemplary bottom view of test cartridge 700 is shown in FIG. 7b . Inthis example, test cartridge 700 may include Authentication Chip 718.

An exemplary cartridge movement apparatus 800, for performing reagentwashing and mixing is shown in FIG. 8. In this example, apparatus 800may include a servo motor 802, a rack 804 and pinion 806 gear mechanism,and a permanent magnet 808. Servo motor 802 may be controlled by controlcircuitry, as described below, and may turn pinion gear 806, causingmovement of rack 804 and thus, movement of permanent magnet 808, whichis attached to rack 804. Movement of permanent magnet 808 may be used tomove the magnetic particles in cartridge 700. Permanent magnet 808 may,for example, be a neodymium alloy magnet. Using rack and pinionmechanism to move micromagnetic beads inside the cartridge to providemanipulation and movement of micromagnetic beads to achieve mixing andwashing actions inside the cartridge reservoirs and for transporting the“washed” micromagnetic beads to the detection reservoir.

An exemplary embodiment of fluorescence detection apparatus 900 is shownin FIG. 9. As shown in this example, Multispectral photodiode chips 902and excitation LEDs 904 may be arranged adjacent to the detection regionin chamber 710, which contains the washed magnetic particles withattached IgA, IgM, and IgG and fluorescent compound. Excitation LEDs904, as controlled by control circuitry, as described below, mayilluminate the contents of chamber 710, causing excitation offluorescent compounds and emission of light from the fluorescentcompounds. Multispectral photodiode chips 902 may receive and detect theemission spectrum of light emitted from the fluorescent compounds, whichmay be analyzed, for example, by optical sensing circuitry and/orcomputing devices, as described herein, to determine the presence orabsence of COVID-19 infection, antibodies, etc.

An exemplary block diagram of a test cartridge 1000 is shown in FIG. 10.In this example, test cartridge 1000 is a disposable sample cartridgemade, for example, from clear glass or silica, transparent acrylic,other plastic, or other transparent material. Cartridge 1000 may includemixer chamber 1002, mixer reservoir 1003, wash chamber 1004, washreservoir 1005, optional amplification chamber 1006, optionalamplification reservoir 1007, detection reservoir 1010, andauthentication chip 1008. Reservoirs 1003, 1005, and 1007 may containsolvents, reagents, buffers, etc., in hermitically sealed blisters forstorage. Mixer reservoir 1003 may be pre-filled with the necessaryreagent solvent and micromagnetic beads or particles having antigens toantibodies raised against SARS-CoV-2 immobilized on the particles. Apatient saliva sample may be introduced into mixer chamber 1002 duringtesting and the reagent solvent and micromagnetic beads may be moved tomixer chamber 1002. Antibodies present in the sample will bind to theantigens immobilized onto magnetic particles. Mixing may be facilitatedby movement of the magnetic particles. The magnetic particles may bemoved by application of electric current by testing device circuitry tomagnetic coils or by movement of cartridge 1000 relative to a magneticfield produced by magnetic coils or by a permanent magnet.

Wash reservoir 1005 may be pre-filled with buffer agent. Micromagneticbeads may be magnetically transported from mixer chamber 1002 to washchamber 1004, and buffer agent may be moved to wash chamber 1004. Inwash chamber 1004, the micromagnetic beads may be “cleaned” to ensureonly bound analytes are detected. Optional amplification reservoir 1007may be prefilled with enzymatic amplification agents or buffer.Micromagnetic beads may be magnetically transported from wash chamber1004 to amplification chamber 1006, and enzymatic amplification agentsor buffer may be moved to amplification chamber 1006. In amplificationchamber 1006, optional enzymatic amplification may be performed.Detection reservoir 1010 may be pre-filled with buffer agent.Micromagnetic beads may be magnetically transported from wash reservoir1004 or optional amplification chamber 1006 to detection reservoir 1010,where, for infected patients, fluorescence from micromagnetic beads maybe detected by way of illuminating LEDs and multi-spectrum photodiodes,as described herein.

An exemplary block diagram of a testing device 1100 is shown in FIG. 11.In this example, testing device 1100 may include power supply 1102, userinterface 1104, such as an LED display and membrane switch buttons,communication interface 1106, such as a USB port or wirelesscommunications adapter, microcontroller 1108, servo motor drivers 1110,detection and authentication circuitry 1112, LED driver 1114, sampleexcitation LEDs 1116, servo motor, rack and pinion, and magnet assembly1118, light detector 1120, and insertable sample cartridge(s) 1122.Power supply 1102 may provide electrical power to the other componentsof testing device 1100 and may include batteries or other electrical andelectronic components, such as voltage regulators to provide differentvoltage supply levels as needed. User interface 1104, may include, forexample, an LED display and membrane switch buttons, or other displayand/or input/output devices. Embodiments may include otherconfigurations of front panel, as well as other display devices, such asLCD displays, numeric displays, etc., which may display additionalinformation, such as concentration, amount, percentage, etc., ofantibodies, fluorescent indicator, threshold levels, etc.

Communication interface 1106, may include, for example a USB port orwireless communications adapter, such as Wi-Fi, Bluetooth, cellulardata, or other wireless communications technique. Microcontroller 1108may include one or more processors, memory, input/output circuitry, andother circuitry to control the operation of testing device 1100 and toprovide interfacing to external computers for data transfer ofinformation including test results, patient information, etc., via USBport or wireless communications adapter and to provide interfacing tothe authentication chip on the sample cartridge. Servo motor drivers1110 may include electronic circuitry to provide electrical current todrive the operation of servo motor 1118 as controlled by microcontroller1108. Detection and authentication circuitry 1112 may include circuitryto interface microcontroller 1108 with authentication chip 718, 1008,shown in FIGS. 7 and 10. Such authentication chips may includeauthentication circuitry, identification circuitry, data storagecircuitry, etc., and may identify the type of chip, the type of testingbeing performed, the patient being tested, etc. Detection andauthentication circuitry 1112 may allow microcontroller 1108 to accessthis information, allowing configuration of testing device 1100 basedthereupon, for example, allowing the device to work in “live” or“testing” mode as required.

LED driver 1114 may include electronic circuitry to provide electricalcurrent to drive the operation of sample excitation LEDs 1116, ascontrolled by microcontroller 1108. Sample excitation LEDs 1116 mayprovide light to excite the fluorescent compounds in the sample undertest, as described herein. For example, sample excitation LEDs may emitlight for excitation of fluorescent compounds at 500 nm wavelength, orother suitable wavelength for excitation of the fluorescent compounds.Multiple LEDs may be provided to increase the intensity, as well as toachieve a more uniform illumination of the sample. Servo motor, rack andpinion, and magnet assembly 1118 may include, for example, the apparatusshown in FIG. 8, and described in reference thereto. Light detector 1120may include electronic circuitry to detect light emitted by the excitedfluorescent compounds in the sample under test, as described herein.Light detector 1120 may, for example, include multispectral photodiodeschips, such as the AMS® multi-channel AS7265x chipset, or other suitablelight detectors. Sample cartridge 1120 may include, for example, a testcartridge 700 similar to that shown in FIGS. 7a, 7b , 9, 10, etc.

An exemplary front view of a front panel 1200 of testing device 1100 isshown in FIG. 12a . In this example, front panel 1200 may include testresult indicators 1202, test in progress indicator 1204, and operationswitch 1206. In this example, test result indicators 1202 may includethree by three LEDs (red, yellow, green) to provide intuitive testresult based on predetermined threshold levels for IgA, IgM, and IgG. Inembodiments, only one color (green, yellow, or red) will result for eachantibody. For example, a green light may indicate the user sample ispositive for the respective antibody, a red light may indicate the userdoes not have the respective antibody in high enough quantities to bedetected by the device, a yellow light may indicate an indeterminateresult. Research may show (for example, see FIG. 16 and Table 1) thatIgA and IgM are produced and persist in early stages of the disease(Days 0-14), and IgG is produced starting on Day 0 of the disease andlevels plateau after Day 20 (Guo, et al., 2020).

TABLE 1 IgA early stage IgM early stage IgA + IgM early stage IgA + IgGearly stage IgM + IgG early stage IgA + IgM + IgG early stage IgG latestage

This may roughly imply that a positive for IgA or IgM means the patientis in early stages of infection, and the presence of IgG alone means thepatient is in later stages of infection. Thus, embodiments may detectwhich of the patients has a mounted immune response to the virus. As thetest will be able to detect which antibodies bind to the spike protein,which is the protein that binds to the ACE receptors in the lungs tocause infection, this test may be able to detect which patients haveraise neutralizing antibodies toward the virus.

Returning to FIG. 12a , test in progress indicator 1204 may include astatus LED indicating the test in progress. Operation switch 1206 mayinclude a membrane switch for an operator to start the test process.Embodiments may include other configurations of front panel, as well asother display devices, such as LCD displays, numeric displays, etc.,which may display additional information, such as concentration, amount,percentage, etc., of antibodies, fluorescent indicator, thresholdlevels, etc.

An exemplary rear view of a front panel 1200 of testing device 1100 isshown in FIG. 12b . In this example, front panel 1200 may include aplurality of spring-backed actuators 1208 in a closable door portion1210 of front panel 1200 to apply pressure, when the door is closed, tothe reservoir blisters and move the reagents, solvents, and buffers,etc. into their associated chambers.

An exemplary internal view of testing device 1100 is shown in FIGS. 13aand 13b . In this example, USB-B connector 1302, authentication chipreader connector 1304, and cartridge holder tray 1306 are shown.

An exemplary external view of testing device 1100 is shown in FIG. 17.In this example, after a sample cartridge is inserted into testingdevice 1100, door 1210 may be closed and a latch 1702 may be activated,preventing removal of the cartridge during the test. When door 1210 isclosed, spring-backed actuators 1208, shown in FIG. 12b , may applypressure to the reservoir blisters and move the reagents, solvents, andbuffers, etc. into their associated chambers.

An exemplary testing system 1400 is shown in FIG. 14. System 1400 mayinclude testing device 1402, computing device 1404, network 1406, andcloud computing system 1408. Testing device 1402 may be a stand-alone orintegrated device and may include testing hardware 1410, controlcircuitry 1412, and communications circuitry 1414. Testing hardware 1410may include the components shown in FIGS. 3, 4, and/or 5, as describedabove, and that performs the testing functions described above. Testinghardware 1410 may include components to interface with an inserted testcartridge 1420, which may include test components 1422, such as amicrofluidic silica chip with etched capillaries and chambers, andcircuitry 1424, which may include authentication circuitry,identification circuitry, data storage circuitry, etc. Testing hardware1410 may further include components such as circuitry to apply, and tocontrol the application of, electric current to magnetic coils that maybe formed on cartridge 1420 or that may be present in the testing device1402 and may be adjacent to or in the vicinity of cartridge 1420.Control circuitry 1412 may include control logic, controller, orprocessor circuitry to control performance of the physical, optical,electrical, and computing processes involved in operating testing device1402, such as controlling the electric current in the magnetic coils,and in performing the functions described above. Communicationscircuitry 1414 may include circuitry to provide wired and/or wirelesscommunications with one or more external or integrated devices, such ascomputing device 1404. In embodiments, testing device 1402 may alsoinclude interface 1415, which may include indicator or displaycomponents for direct display of test results from testing device 1402and/or buttons, etc., for direct entry of information into testingdevice 1402.

Computing device 1404 may be an integrated or stand-alone device, suchas a smartphone, tablet computer, laptop computer, personal computer,workstation computer, cloud computing service, etc., to communicate withtesting device 1402. Computing device 1404 may provide processing andanalysis of data received from testing device 1402, as well ascommunications with testing device 1402 and with cloud computing system1408 vis network 1406. In embodiments, computing device 1404 may alsoinclude indicator or interface displays (not shown) for display of testresults from testing device 1402 and/or entry of information intotesting device 1402. Network 1406 may be any public or proprietary LANor WAN, including, but not limited to the Internet, carrier network,wireless network, etc. Cloud computing system 1408 may provide on-demandavailability of computer system resources, such as database storage 1416and computing power/data analysis 1418, which is typically implementedin data centers available to many users over the Internet.

In embodiments, the optical analysis may result in imagingrepresentative of sample 402. The resulting imaging, using machinelearning techniques, may be used to detect protein structure geometry(Daaboul, G. G., et al. 2017). Then, combined inputs from chemicallab-on-chip sensors and the optical sensors, both included in testinghardware 1410 may be analyzed 1418, for example, using cloud computingsystem 1408, using both commonly available and proprietary ArtificialIntelligence/Machine Learning (AWL) architectures, such as a DeepCognitive Neural Network (DCNN), such as that described in U.S. PatentApplication Publication No. 2019/0156189, published May 23, 2019, whichis hereby incorporated by reference herein. Machine learning algorithmsmay be trained to detect the coronavirus family of viruses, and overtime, using additional sample data, will become more effective atidentifying different strains of the virus.

In embodiments, the machine algorithms may be further trained to detectother classes of viruses and specific strains of viruses or otherpathogens, or trained for detection of neurodegenerative diseasemarkers. The computing device 1404 computational platform may interfacewith a cloud computing platform 1408, opening up applications usinganonymized patient and third party data.

An exemplary block diagram of a computing device 1500, in whichprocesses involved in the embodiments described herein, such ascomputing device 1406 or cloud computing system 1408, may beimplemented, is shown in FIG. 15. Computing device 1500 may beimplemented using one or more programmed general-purpose computersystems, such as embedded processors, systems on a chip, personalcomputers, workstations, server systems, and minicomputers or mainframecomputers, or in distributed, networked computing environments.Computing device 1500 may include one or more processors (CPUs)1502A-1502N, input/output circuitry 1504, network adapter 1506, andmemory 1508. CPUs 1502A-1502N execute program instructions in order tocarry out the functions of the present communications systems andmethods. Typically, CPUs 1502A-1502N are one or more microprocessors,such as an INTEL CORE® processor. FIG. 15 illustrates an embodiment inwhich computing device 1500 is implemented as a single multi-processorcomputer system, in which multiple processors 1502A-1502N share systemresources, such as memory 1508, input/output circuitry 1504, and networkadapter 1506. However, the present communications systems and methodsalso include embodiments in which computing device 1500 is implementedas a plurality of networked computer systems, which may besingle-processor computer systems, multi-processor computer systems, ora mix thereof.

Input/output circuitry 1504 provides the capability to input data to, oroutput data from, computing device 1500. For example, input/outputcircuitry may include input devices, such as keyboards, mice, touchpads,trackballs, scanners, analog to digital converters, etc., outputdevices, such as video adapters, monitors, printers, etc., andinput/output devices, such as, modems, etc. Network adapter 1506interfaces device 1500 with a network 1510. Network 1510 may be anypublic or proprietary LAN or WAN, including, but not limited to theInternet.

Memory 1508 stores program instructions that are executed by, and datathat are used and processed by, CPU 1502 to perform the functions ofcomputing device 1500. Memory 1508 may include, for example, electronicmemory devices, such as random-access memory (RAM), read-only memory(ROM), programmable read-only memory (PROM), electrically erasableprogrammable read-only memory (EEPROM), flash memory, etc., andelectro-mechanical memory, such as magnetic disk drives, tape drives,optical disk drives, etc., which may use an integrated drive electronics(IDE) interface, or a variation or enhancement thereof, such as enhancedIDE (EIDE) or ultra-direct memory access (UDMA), or a small computersystem interface (SCSI) based interface, or a variation or enhancementthereof, such as fast-SCSI, wide-SCSI, fast and wide-SCSI, etc., orSerial Advanced Technology Attachment (SATA), or a variation orenhancement thereof, or a fiber channel-arbitrated loop (FC-AL)interface.

The contents of memory 1508 may vary depending upon the function thatcomputing device 1500 is programmed to perform. In the example shown inFIG. 15, exemplary memory contents are shown representing routines anddata for embodiments of the processes described above. However, one ofskill in the art would recognize that these routines, along with thememory contents related to those routines, may not be included on onesystem or device, but rather may be distributed among a plurality ofsystems or devices, based on well-known engineering considerations. Thepresent systems and methods may include any and all such arrangements.

In the example shown in FIG. 15, memory 1508 may include, in the case ofa testing device 1402, testing control data and routines 1512, testinganalysis data and routines 1514, in the case of a computing device 1404,data analysis data and routines 1518, and in the case of a cloudcomputing system 1408, database data and routines 1520, and dataanalysis data and routines 1522 and operating system 1524. Testingcontrol data and routines 1512 may include software routines to controlperformance of the physical, optical, electrical, and computingprocesses involved in operating testing device 1402, as well as dataobtained from such testing, as described above. Testing analysis dataand routines 1514 may include software routines to perform initialanalysis and derivation of data obtained from testing, as describedabove. Data analysis data and routines 1514 may include, which mayinclude software routines to perform processing and analysis of datareceived from testing device 1402, as described above.Authentication/matching routines 1514 may include modular proximity testroutines 1518, which may include software routines to perform modularproximity testing on received authentication data, as described above.Database data and routines 1520, may include software routines toprovide database storage of data on cloud computing system 1408, asdescribed above. Data analysis data and routines 1522 may includesoftware routines to provide computing power/data analysis on cloudcomputing system 1408, as described above. Operating system 1524 mayprovide overall system functionality.

As shown in FIG. 15, the present communications systems and methods mayinclude implementation on a system or systems that providemulti-processor, multi-tasking, multi-process, and/or multi-threadcomputing, as well as implementation on systems that provide only singleprocessor, single thread computing. Multi-processor computing involvesperforming computing using more than one processor. Multi-taskingcomputing involves performing computing using more than one operatingsystem task. A task is an operating system concept that refers to thecombination of a program being executed and bookkeeping information usedby the operating system. Whenever a program is executed, the operatingsystem creates a new task for it. The task is like an envelope for theprogram in that it identifies the program with a task number andattaches other bookkeeping information to it. Many operating systems,including Linux, UNIX®, OS/2®, and Windows®, are capable of running manytasks at the same time and are called multitasking operating systems.Multi-tasking is the ability of an operating system to execute more thanone executable at the same time. Each executable is running in its ownaddress space, meaning that the executables have no way to share any oftheir memory. This has advantages, because it is impossible for anyprogram to damage the execution of any of the other programs running onthe system. However, the programs have no way to exchange anyinformation except through the operating system (or by reading filesstored on the file system). Multi-process computing is similar tomulti-tasking computing, as the terms task and process are often usedinterchangeably, although some operating systems make a distinctionbetween the two.

The present invention may be a system, a method, and/or a computerprogram product at any possible technical detail level of integration.The computer program product may include a computer readable storagemedium (or media) having computer readable program instructions thereonfor causing a processor to carry out aspects of the present invention.The computer readable storage medium can be a tangible device that canretain and store instructions for use by an instruction executiondevice.

The computer readable storage medium may be, for example, but is notlimited to, an electronic storage device, a magnetic storage device, anoptical storage device, an electromagnetic storage device, asemiconductor storage device, or any suitable combination of theforegoing. A non-exhaustive list of more specific examples of thecomputer readable storage medium includes the following: a portablecomputer diskette, a hard disk, a random access memory (RAM), aread-only memory (ROM), an erasable programmable read-only memory (EPROMor Flash memory), a static random access memory (SRAM), a portablecompact disc read-only memory (CD-ROM), a digital versatile disk (DVD),a memory stick, a floppy disk, a mechanically encoded device such aspunch-cards or raised structures in a groove having instructionsrecorded thereon, and any suitable combination of the foregoing. Acomputer readable storage medium, as used herein, is not to be construedas being transitory signals per se, such as radio waves or other freelypropagating electromagnetic waves, electromagnetic waves propagatingthrough a waveguide or other transmission media (e.g., light pulsespassing through a fiber-optic cable), or electrical signals transmittedthrough a wire.

Computer readable program instructions described herein can bedownloaded to respective computing/processing devices from a computerreadable storage medium or to an external computer or external storagedevice via a network, for example, the Internet, a local area network, awide area network and/or a wireless network. The network may comprisecopper transmission cables, optical transmission fibers, wirelesstransmission, routers, firewalls, switches, gateway computers, and/oredge servers. A network adapter card or network interface in eachcomputing/processing device receives computer readable programinstructions from the network and forwards the computer readable programinstructions for storage in a computer readable storage medium withinthe respective computing/processing device.

Computer readable program instructions for carrying out operations ofthe present invention may be assembler instructions,instruction-set-architecture (ISA) instructions, machine instructions,machine dependent instructions, microcode, firmware instructions,state-setting data, configuration data for integrated circuitry, oreither source code or object code written in any combination of one ormore programming languages, including an object oriented programminglanguage such as Smalltalk, C++, or the like, and procedural programminglanguages, such as the “C” programming language or similar programminglanguages. The computer readable program instructions may executeentirely on the user's computer, partly on the user's computer, as astand-alone software package, partly on the user's computer and partlyon a remote computer or entirely on the remote computer or server. Inthe latter scenario, the remote computer may be connected to the user'scomputer through any type of network, including a local area network(LAN) or a wide area network (WAN), or the connection may be made to anexternal computer (for example, through the Internet using an InternetService Provider). In some embodiments, electronic circuitry including,for example, programmable logic circuitry, field-programmable gatearrays (FPGA), or programmable logic arrays (PLA) may execute thecomputer readable program instructions by utilizing state information ofthe computer readable program instructions to personalize the electroniccircuitry, in order to perform aspects of the present invention.

Aspects of the present invention are described herein with reference toflowchart illustrations and/or block diagrams of methods, apparatus(systems), and computer program products according to embodiments of theinvention. It will be understood that each block of the flowchartillustrations and/or block diagrams, and combinations of blocks in theflowchart illustrations and/or block diagrams, can be implemented bycomputer readable program instructions.

These computer readable program instructions may be provided to aprocessor of a general-purpose computer, special purpose computer, orother programmable data processing apparatus to produce a machine, suchthat the instructions, which execute via the processor of the computeror other programmable data processing apparatus, create means forimplementing the functions/acts specified in the flowchart and/or blockdiagram block or blocks. These computer readable program instructionsmay also be stored in a computer readable storage medium that can directa computer, a programmable data processing apparatus, and/or otherdevices to function in a particular manner, such that the computerreadable storage medium having instructions stored therein comprises anarticle of manufacture including instructions which implement aspects ofthe function/act specified in the flowchart and/or block diagram blockor blocks.

The computer readable program instructions may also be loaded onto acomputer, other programmable data processing apparatus, or other deviceto cause a series of operational steps to be performed on the computer,other programmable apparatus or other device to produce a computerimplemented process, such that the instructions which execute on thecomputer, other programmable apparatus, or other device implement thefunctions/acts specified in the flowchart and/or block diagram block orblocks.

The flowchart and block diagrams in the Figures illustrate thearchitecture, functionality, and operation of possible implementationsof systems, methods, and computer program products according to variousembodiments of the present invention. In this regard, each block in theflowchart or block diagrams may represent a module, segment, or portionof instructions, which comprises one or more executable instructions forimplementing the specified logical function(s). In some alternativeimplementations, the functions noted in the blocks may occur out of theorder noted in the Figures. For example, two blocks shown in successionmay, in fact, be executed substantially concurrently, or the blocks maysometimes be executed in the reverse order, depending upon thefunctionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts or carry out combinations of special purpose hardwareand computer instructions.

Although specific embodiments of the present invention have beendescribed, it will be understood by those of skill in the art that thereare other embodiments that are equivalent to the described embodiments.Accordingly, it is to be understood that the invention is not to belimited by the specific illustrated embodiments, but only by the scopeof the appended claims.

1. A device for detecting primary antibodies to a pathogen or thepathogen in a person comprising: a cartridge configured to receive atest sample from the person, wherein the test sample is saliva, thecartridge comprising at least one chamber configured to receive the testsample; a first reagent reactive to presence of primary antibodies tothe pathogen or the pathogen, wherein the pathogen is SARS-CoV-2; firstapparatus configured to mix at least the first reagent reactive topresence of the primary antibodies to the pathogen or the pathogen withthe test sample; a second reagent including a fluorescent compoundreactive to presence of the at least one first reagent having reacted topresence of the primary antibodies to the pathogen or the pathogen;second apparatus configured to mix at least one second reagent with thetest sample mixed with the first reagent; and circuitry configured todetermine presence of primary antibodies to the pathogen or the pathogenby detecting reaction of the second reagent by determining fluorescenceof the fluorescent compound.
 2. The device of claim 1, wherein the firstapparatus further comprises a plurality of magnetic particles upon whichat least one antigen to primary antibodies to the pathogen has beenimmobilized, wherein the at least one first reagent comprises the atleast one antigen to primary antibodies that has been immobilized on theplurality of magnetic particles, wherein the antigen is SARS-CoV-2 S1protein.
 3. The device of claim 2, wherein the first apparatus furthercomprises apparatus configured to mix the plurality of magneticparticles with the test sample so as to cause the primary antibodies tothe pathogen to attach to the antigen.
 4. The device of claim 3, whereinthe second apparatus comprises apparatus configured to mix the magneticparticles having the primary antibodies to the pathogen to attachedthereto with the at least one second reagent including a fluorescentcompound.
 5. The device of claim 4, wherein the at least one secondreagent including a fluorescent compound comprises at least onesecondary antibody labeled with a fluorescent compound.
 6. The device ofclaim 1, wherein the circuitry configured to determine presence ofprimary antibodies to the pathogen or the pathogen by detecting reactionof the second reagent by determining fluorescence of the fluorescentcompound comprises a fluorometer.
 7. The device of claim 6, wherein thecircuitry configured to determine presence of primary antibodies to thepathogen or the pathogen by detecting reaction of the second reagent bydetermining fluorescence of the fluorescent compound comprises: a lightsource configured to excite the fluorescent compound with a of light,and an optical sensor configured to detect an emitted spectrum of lightfrom the excited fluorescent compound.
 8. The device of claim 1, furthercomprising display circuitry configured to display an indication ofpresence or absence of primary antibodies to the pathogen. 9-11.(canceled)
 12. The device of claim 5, wherein the secondary antibodiescomprise IgA, IgM, and IgG and each of IgA, IgM, and IgG is labeled witha fluorescent compound having a different light emission spectrum.13-22. (canceled)